CN108548885B - Method for detecting compound arisaema analgesic plaster by two-dimensional liquid chromatography - Google Patents
Method for detecting compound arisaema analgesic plaster by two-dimensional liquid chromatography Download PDFInfo
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Abstract
The invention discloses a method for detecting diester alkaloids in compound arisaema analgesic plaster. The method comprises the following steps: step one, preparing a test solution, namely extracting and removing impurities from a compound arisaema analgesic plaster sample to prepare the test solution; and step two, absorbing the test solution, and determining the diester alkaloid by adopting a two-dimensional liquid chromatography method. The method provided by the invention has better accuracy and stability, effectively improves the separation degree of components to be detected in the compound arisaema analgesic plaster, greatly improves the detection sensitivity, accurately and quantitatively analyzes three diester alkaloids in the preparation, improves the quality control level of the product, and provides theoretical support for clinical medication.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicines, and in particular relates to a method for detecting compound arisaema analgesic ointment by two-dimensional liquid chromatography.
Background
In recent years, with the gradual maturity of commercial two-dimensional liquid chromatography instrument technologies, two-dimensional liquid chromatography combining multiple separation modes, such as center cutting, multi-center cutting, full two-dimensional liquid chromatography, and the like, appears, which can be divided into an online mode and an offline mode, wherein the online two-dimensional liquid chromatography can selectively transfer one-dimensional fractions to a second-dimensional chromatographic column for analysis, and can well solve the problem of limited separation capacity of conventional chromatography.
The compound arisaema analgesic plaster is composed of 12 medicines of arisaema tuber, unprocessed radix aconiti, clove, cinnamon, angelica dahurica, asarum, rhizoma ligustici wallichii, paniculate swallowwort root, frankincense, myrrh, camphor and borneol, is an analgesic prescription which is classically formed by a plurality of traditional Chinese medicines, and has the effects of dispelling cold, removing dampness, promoting blood circulation and relieving pain. The raw radix aconiti is used as a monarch drug in the prescription, has the effects of dispelling wind, removing dampness, warming channels and relieving pain, wherein the diester alkaloid is used as a characteristic active ingredient of the raw radix aconiti, has the effect of relieving pain and has stronger toxicity. Because the compound arisaema analgesic plaster has extremely complex components and contains auxiliary materials such as vaseline, liquid paraffin and the like, the pretreatment method of the sample is complicated, and the existing quality standard of the preparation only adopts an HPLC method to carry out content determination on the hypaconitine in the preparation. Therefore, it is necessary to establish a detection method for simultaneously measuring a plurality of diester alkaloids in a preparation.
Disclosure of Invention
In view of the above, the present invention provides a method for detecting diester alkaloids in compound arisaema analgesic plaster, which is characterized by comprising the following steps:
step one, preparing a test solution, namely extracting and removing impurities from a compound arisaema analgesic plaster sample to prepare the test solution;
and step two, absorbing the test solution, and determining the diester alkaloid by adopting a two-dimensional liquid chromatography method.
Specifically, the two-dimensional liquid chromatography method is carried out by adopting the following method:
a one-dimensional chromatographic method: c18In the first chromatographic column, the mobile phase A is methanol, the mobile phase B is 0.001-0.02% of diethylamine or triethylamine, and the flow rate is 0.05-0.2 mL/min-1Eluting with 40-70% of mobile phase A at the column temperature of 15-20 ℃ for 40min in a gradient manner, wherein the detection wavelength is 230-240 nm, the sample injection amount is 1-3 mu L, and the temperature of a sample plate is 8-20 ℃;
the valve switching method comprises the following steps: the valve switching time is from the initial peak output time to the end of the diester alkaloid;
two-dimensional chromatographic methods: c18A second chromatographic column, wherein the mobile phase A is methanol or acetonitrile, the mobile phase B is 0.005-0.02% phosphoric acid or diethylamine, and the flow rate is 0.5-0.8 mL/min-1And the column temperature is 35-45 ℃, the gradient elution is carried out for 4min by using 30-90% of mobile phase A, and the detection wavelength is 230-240 nm.
Preferably, the two-dimensional liquid chromatography method is performed using the following method:
a one-dimensional chromatographic method: said C is18The first specification of the chromatographic column is 1 × 50mm and 1.7 μm, the mobile phase A is methanol, the mobile phase B is 0.008% DEA, and the flow rate is 0.1 mL/min-1Column temperature 18 ℃; gradient elution, 0-12 min, 40-58% A, 12-25 min, 58-58% A, 25-35 min, 58-65% A, 35-40 min, 65-70% A, 40-42 min, 70-70% A, 42-45 min, 70-100% A, 45-48 min, 100-40% A; the detection wavelength is 235nm, the sample injection amount is 1 mu L, and the temperature of a sample plate is 10 ℃;
the valve switching method comprises the following steps: the valve switching time is from the initial peak output time to the end of the diester alkaloid;
two-dimensional chromatographic methods: said C is18The second specification of the chromatographic column is 3.0 × 100mm and 1.7 μm, the mobile phase A is acetonitrile, the mobile phase B is 0.01% phosphoric acid, and the flow rate is 0.7 mL/min-1The column temperature is 40 ℃; gradient elution, 0-1 min, 30-38% A, 1-3 min, 38-48% A, 3-4 min, 48-90% A, 4-4.2 min and 90-30% A; the detection wavelength was 235 nm.
In particular, said C18Chromatography column one and said C18The second chromatographic column is selected from Waters Xbridge BEH C18A chromatographic column is arranged on the top of the chromatographic column,
further: the extraction and impurity removal in the first step are as follows: adding appropriate amount of methanol into the product, grinding, quantitatively transferring to a measuring flask, ultrasonic treating, diluting with methanol to scale, shaking, filtering, discarding the primary filtrate, collecting the subsequent filtrate, and recovering under reduced pressure to dry; adding hydrochloric acid solution, slightly heating to dissolve the residue, shaking, refrigerating in refrigerator, filtering, collecting the filtrate, placing in a separating funnel, extracting with chloroform, mixing chloroform extractive solutions, dehydrating with anhydrous sodium carbonate, recovering the filtrate under reduced pressure to dryness, dissolving the residue with methanol, metering volume, shaking, and centrifuging.
Specifically, the extraction and impurity removal method comprises the steps of taking 10 tablets of the product, removing the lining cloth, scraping the ointment, taking about 8g of 2 tablets, precisely weighing, placing in a mortar, adding an appropriate amount of methanol, uniformly grinding, quantitatively transferring to a 100mL measuring flask, ultrasonically treating for 1h, placing for 1h, adding methanol to dilute to a scale, uniformly shaking, filtering, discarding primary filtrate, precisely measuring 50mL of subsequent filtrate, and recovering under reduced pressure until the subsequent filtrate is dried. Adding 2% hydrochloric acid solution 100mL precisely, slightly heating to dissolve the residue, shaking, cooling, refrigerating in refrigerator for 1h, filtering, precisely measuring the amount of filtrate 50mL, placing in separating funnel, extracting with chloroform (40, 30mL) in several times, mixing chloroform extractive solutions, dehydrating with anhydrous sodium carbonate, recovering the filtrate under reduced pressure to dryness, dissolving the residue with methanol to constant volume of 2mL, shaking, 14000 r.min-1Centrifuging for 10min to obtain the final product.
Further, the diester alkaloids include, but are not limited to, mesaconine, aconitine and hypaconitine.
The invention provides a method for detecting compound arisaema analgesic plaster, which is characterized in that a two-dimensional liquid chromatography method is adopted to carry out qualitative or quantitative detection on diester alkaloids.
Specifically, the two-dimensional liquid chromatography method includes one-dimensional chromatography and two-dimensional chromatography; wherein, the one-dimensional chromatographic analysis selects diethylamine or triethylamine with methanol-0.001-0.02% as a mobile phase system.
Further, the two-dimensional chromatographic analysis selects methanol or acetonitrile-0.005-0.02% phosphoric acid or diethylamine as a mobile phase system.
Preferably, methanol-0.008% DEA is used as the mobile phase system for the one-dimensional chromatographic analysis, and acetonitrile-0.01% phosphoric acid is used as the mobile phase system for the two-dimensional chromatographic analysis.
The extraction and separation method of the invention has the following advantages:
(1) the on-line two-dimensional liquid chromatography can selectively transfer the one-dimensional fraction to a second-dimensional chromatographic column for analysis, can well solve the problem of limited separation capacity of the conventional chromatography, and enables mesaconitine and aconitine which are difficult to detect in the conventional detection method to be detected.
(2) The method provided by the invention has low requirement on impurity removal in the sample pretreatment, so that the sample pretreatment method can be simplified.
(3) Under the detection condition provided by the invention, the obvious mesaconine and aconitine can be detected, the interference of impurity components is greatly eliminated, the separation effect is improved, and the detection sensitivity is improved.
(4) The method for detecting the compound arisaema analgesic plaster has good repeatability, can effectively separate the components to be detected, has stable and accurate measurement result, and can be applied to the simultaneous quantification of a plurality of diester alkaloid components in the preparation. The method provided by the invention effectively improves the separation degree of components to be detected in the compound arisaema analgesic plaster, greatly improves the detection sensitivity, improves the quality control level of the product, and provides theoretical support for clinical medication.
(5) In the preparation process of the sample solution, the processes of extracting and removing impurities of the compound arisaema analgesic plaster can reduce the impurities to the minimum and improve the subsequent separation effect.
(6) The detection method of the invention optimizes the traditional liquid chromatogram, improves the peak capacity of the system, enhances the separation capability of the chromatographic system and quickly separates the complex components of the preparation. Due to the establishment of the online analysis mode, the error of manual operation is eliminated, the analysis efficiency of the sample is accelerated, the accuracy and the stability of the analysis result of the sample are ensured, and the problem that the trace components in the compound arisaema analgesic plaster are difficult to quantify is effectively solved.
Drawings
FIG. 1 is a schematic diagram of the configuration and connection of an ultra-high performance two-dimensional liquid chromatography system;
FIG. 2 is a full-wavelength scanning spectrum of neoaconitine (A), aconitine (B) and hypaconitine (C) reference substances;
FIG. 3 is a two-dimensional UHPLC separation spectrum of methanol-0.008% DEA/acetonitrile-0.02% DEA;
FIG. 4 is a two-dimensional UHPLC separation spectrum of methanol-0.008% DEA/acetonitrile-0.01% phosphoric acid;
FIG. 5 is an ultra-efficient two-dimensional liquid phase separation spectrum of the mixed control (A: one-dimensional UHPLC separation spectrum; B: two-dimensional UHPLC separation spectrum);
FIG. 6 is an ultra-high-efficiency two-dimensional liquid phase separation spectrum of compound rhizoma arisaematis analgesic plaster (A: one-dimensional UHPLC separation spectrum; B: two-dimensional UHPLC separation spectrum);
FIG. 7 is an ultra-efficient two-dimensional liquid phase separation spectrogram of the unprocessed radix aconiti carmichaeli negative preparation (A: one-dimensional UHPLC separation spectrogram; B: two-dimensional UHPLC separation spectrogram);
FIG. 8 is a one-dimensional detection spectrum of compound rhizoma arisaematis analgesic plaster (the chromatographic peak with retention time of 40.308 is hypaconitine); FIG. 9 is the mixed reference solution spectrum of FIG. 8 under the same detection conditions (three peaks from left to right are mesaconine, aconitine and hypaconitine, respectively).
In the above drawings: 1: mesaconine, 2: aconitine, 3: hypaconitine.
Detailed Description
As mentioned above, the present invention aims to provide a method for detecting diester alkaloids in compound arisaema analgesic plaster.
An experimental instrument: agilent 1290Infinity II two-dimensional liquid chromatography system (including 1290Infinity II high speed pump, 1290Infinity II autosampler, 1290Infinity II column oven, 1290Infinity II diode array detector, 1290Infinity II binary pump for second dimension analysis, 1290Infinity II diode array detector for second dimension analysis, external valve drive), BUCHI R-3 rotary evaporator, KH-300DB type numerically controlled ultrasonic cleaner, ME303E type electronic balance, XS205DU type electronic analytical balance.
Reagents and reagents: aconitine (China food and drug testing institute, batch No. 110798-201609), aconitine (China food and drug testing institute, batch No. 110720-201111), neoaconitine (China food and drug testing institute, batch No. 110799-201307), compound arisaema analgesic paste (Jiangsu Kangyuan sunshine pharmaceutical Co., Ltd., batch No. 160510, 170707, 160609, 160713, 160708, 161126, 160720, 170621, 170626, 170218), acetonitrile (Tedia, batch No. 16045019), methanol (Tedia, batch No. 17095089), anhydrous methanol (national drug group chemical reagent Co., Ltd., batch No. 20151214), chloroform (Nanjing chemical reagent Co., Ltd., batch No. 170424921D), anhydrous sodium carbonate (national drug group chemical reagent Co., Ltd., batch No. 20130513), diethylamine (national drug group chemical reagent Co., Ltd., batch No. 20161214), phosphoric acid (national drug reagent Co., batch No. 13072211046-Nanjing chemical reagent Co., Ltd., batch No. 13072211046), The water is ultrapure water.
The method is carried out according to conventional conditions or conditions recommended by manufacturers, and the raw materials, reagents or instruments used by the method are conventional products which can be obtained commercially.
It is specifically noted that similar alternatives and modifications will be apparent to those skilled in the art, which are also intended to be included within the present invention. It will be apparent to those skilled in the art that the techniques of the present invention may be implemented and applied by modifying or appropriately combining the methods and applications described herein without departing from the spirit, scope, and content of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention.
The following will be described specifically with reference to the experimental contents.
Method for detecting diester alkaloid of compound arisaema analgesic plaster
1 preparation of control solutions
Weighing appropriate amount of neoaconitine, aconitine and hypaconitine, respectively, precisely weighing, placing in 10mL measuring flask, dissolving with chromatographic methanol, and fixing to desired volume to obtain concentrations of 0.990 mg/mL-1、0.820mg·mL-1And 1.370 mg. multidot.mL-1Control stock solution of (4).
2 preparation of test solution
Taking 10 tablets of the product, removing the lining cloth, scraping the ointment, taking about 8g corresponding to 2 tablets, precisely weighing, placing in a mortar, adding a proper amount of methanol, uniformly grinding, quantitatively transferring to a 100mL measuring flask, ultrasonically treating for 1h, placing for 1h, adding methanol to dilute to scale, uniformly shaking, filtering, discarding the primary filtrate, precisely taking 50mL of the subsequent filtrate, and recovering under reduced pressure to dryness. Adding 2% hydrochloric acid solution 100mL precisely, slightly heating to dissolve the residue, shaking, cooling, refrigerating in refrigerator for 1h, filtering, precisely measuring the amount of filtrate 50mL, placing in separating funnel, extracting with chloroform (40, 30mL) in several times, mixing chloroform extractive solutions, dehydrating with anhydrous sodium carbonate, recovering the filtrate under reduced pressure to dryness, dissolving the residue with methanol to constant volume of 2mL, shaking, 14000 r.min-1Centrifuging for 10min to obtain the final product.
3 chromatographic conditions
The configuration and connection of the ultra-high performance two-dimensional liquid chromatography system are shown in fig. 1, and the conditions of the two-dimensional liquid chromatography detection are specifically as follows:
a one-dimensional chromatographic method: waters Xbridge BEH C18(2.1X 50mm, 1.7 μm) column chromatography, mobile phase A methanol, B0.008% DEA, flow rate 0.1mL min-1Gradient elution is carried out at the column temperature of 18 ℃, the gradient elution is carried out for 0-12 min, 40-58% of A, 12-25 min, 58-58% of A, 25-35 min, 58-65% of A, 35-40 min, 65-70% of A, 40-42 min, 70-70% of A, 42-45 min, 70-100% of A, 45-48 min and 100-40% of A; the detection wavelength is 235nm, the sample injection amount is 1 mu L, and the temperature of a sample plate is 10 ℃;
the valve switching method comprises the following steps: the valve switching time was from the initial peak time to the end of the three diester alkaloids, for a total of 3 fragments, and the quantitative loop volume was 80 μ L.
Two-dimensional chromatographic methods: waters Xbridge BEH C18(3.0X 100mm, 1.7 μm) column chromatography with mobile phase A of acetonitrile and B of 0.01% phosphoric acid at a flow rate of 0.7 mL/min-1And the column temperature is 40 ℃, gradient elution is carried out for 0-1 min, 30-38% of A, 1-3 min, 38-48% of A, 3-4 min, 48-90% of A, 4-4.2 min, 90-30% of A, and the detection wavelength is 235 nm.
4 results of detection
4.1 detection wavelength selection
In order to select a proper detection wavelength, the mixed reference substance solution is subjected to full-wavelength scanning in the experiment, and the result is shown in fig. 2, and the maximum absorption wavelengths of mesaconitine, aconitine and hypaconitine are all about 235 nm.
4.2 detection wavelength selection
In order to ensure that the target compound can be completely cut into the second dimension for analysis, systems such as phosphate-DEA buffer salt, DEA (diethylamine), triethylamine and the like with different proportions are tried on the selection of the water phase of the one-dimensional chromatographic system, preferably a methanol-0.008% DEA system, and the peak width of the target compound is small under the system, so that the center cutting operation is convenient to carry out. In terms of the selection of the second-dimension chromatographic conditions, in order to ensure that the collected target components can be completely analyzed without interfering the analysis of the next target compound, the organic phase is preferably changed into acetonitrile with stronger elution capacity, so that the analysis time can be effectively shortened. In order to eliminate the interference of other impurities on the peak separation of the target compound, 0.02% DEA and 0.01% phosphoric acid were tried on the selection of the water phase, and the separation effect of the two-dimensional mobile phase system is shown in FIGS. 3 and 4 (1: mesaconine, 2: aconitine, 3: hypaconitine; peaks 1, 2, 3 in other figures are all marked by the three substances). By comparison, the acetonitrile-0.01% phosphoric acid mobile phase system can improve the peak shape and the separation degree of the target compound and eliminate the interference of other impurity components. Therefore, methanol-0.008% DEA is preferably used as a one-dimensional mobile phase system, and acetonitrile-0.01% phosphoric acid is preferably used as a two-dimensional mobile phase system.
4.3 specificity test
Accurately weighing the radix aconiti-free negative preparation, and preparing a solution to be tested of the negative preparation according to the method under item 2. The 2D-UHPLC separation spectra of the reference substance, sample preparation and negative preparation are shown in FIGS. 5-7 according to the analysis of sample injection under the condition of "3" chromatography. As can be seen from the negative preparation chromatogram, the negative preparation has no interference to the measurement of the sample, and the method has good specificity.
4.4 Linear relationship
Respectively taking appropriate amount of control stock solution under item "1", and preparing with chromatographic methanol to give neoaconitine, aconitine and hypaconitine with concentrations of 19.80 μ g/mL-1、16.39μg·mL-1、27.44μg·mL-1Mixed control solution of (4). Adopting stepwise dilution method to obtain mixed standard solution with series concentration, performing sample injection analysis according to chromatography condition of '3', recording peak area of each chromatographic peak, and determining sample concentration (μ g. mL)-1) The standard curve is plotted with the abscissa (x) and the peak area as the ordinate (y), and the regression equation is calculated. The results are shown in Table 1. The results show that the linear relationship of mesaconitine, aconitine and hypaconitine is good in the selected concentration range.
TABLE 1 Linear regression equation, correlation coefficient and Linear Range investigation
4.5 precision test
Taking the mixed reference substance solution, continuously injecting a sample for 6 needles, recording the peak area of each chromatographic peak, and calculating the RSD value, wherein the result is shown in Table 2. Indicating that the precision of the instrument is good.
4.6 stability test
Taking mixed reference solution and test solution (batch: 170218), injecting samples at 0, 2, 4, 8, 10, 12, 18 and 24h after preparation, recording peak areas of components to be detected, and calculating RSD value, wherein the results are shown in Table 2. The control and test solutions were stable for 24 h.
4.7 repeatability test
Taking 6 parts of compound arisaema analgesic plaster preparation (batch: 170218) of the same batch, each part is about 8g, precisely weighing, preparing a test solution according to the method under item 2, parallelly operating 6 parts, measuring according to the chromatographic condition under item 3, recording the peak area of each component to be measured, and calculating the RSD value of the content, wherein the result is shown in table 2. The results show that the RSD% of the contents of the mesaconine, the aconitine and the hypaconitine are respectively 3.31%, 2.95% and 1.99%, and the content of the components to be measured of the sample is 10 mu g according to the 'Chinese pharmacopoeia' of 2015 edition-1The specification of an acceptable range of RSD of 6% "indicates good reproducibility of the method.
TABLE 2 results of precision, stability and reproducibility test (n ═ 6)
4.8 sample recovery test
6 portions of sample (batch: 170218) with known content, 4g each, are precisely weighed, put into a mortar, added with a proper amount of mixed reference solution, prepared into a test solution according to the method under item 2, the content is measured, and the recovery rate is calculated, and the result is shown in Table 3. The results show that the average recovery rates of the mesaconitine, the aconitine and the hypaconitine are 83.86 percent, 82.49 percent and 88.84 percent respectively, which meet the requirements of 2015 edition Chinese pharmacopoeia on the content of the component to be measured of a sample of 10 mu g-1The sample recovery rate limit should be within the range of 80% to 115%.
TABLE 3 sample recovery of mesaconine, aconitine and hypaconitine (n ═ 6)
4.9 Multi-batch sample assay
Get 10A plaster preparation is prepared by preparing sample solution according to the method of item 2, and measuring the content of mesaconitine, aconitine and hypaconitine according to the chromatographic condition of item 3. Meanwhile, 10 batches of samples are taken, and the content of hypaconitine is determined by an HPLC method in the current standard, and the result is shown in Table 4. The results show that the contents of the aconitine in the two detection methods are basically consistent, and both the two detection methods meet the existing quality standards, and the content of aconitine in the product is 6-12 mu g.g-1"indicates that the method has better applicability.
TABLE 410 measurement of Sinaconitine, aconitine and hypaconitine content in lots of samples (n ═ 3)
An analysis method for simultaneously measuring three diester alkaloids of mesaconine, aconitine and hypaconitine in a compound arisaema analgesic ointment is established based on an online center cutting ultra-high performance two-dimensional liquid chromatography separation technology. Because the preparation has complex components and contains auxiliary materials such as vaseline, liquid paraffin and the like, the interference on target components is serious, and only the hypaconitine is detected under the one-dimensional UHPLC detection condition (see a figure 8 and a figure 9). Therefore, in the experiment, a contrast retention time comparison method is adopted, the peak fragment with the same retention time as the contrast in the preparation is cut into the two-dimensional chromatogram, and the finding shows that the obvious new aconitine and aconitine can be detected in the two-dimensional UHPLC chromatogram, thereby greatly eliminating the interference of impurity components, improving the separation effect and improving the detection sensitivity. The methodological investigation result shows that the method has good repeatability, can effectively separate the components to be measured, has stable and accurate measurement result, and can be applied to the simultaneous quantification of the three diester alkaloids in the preparation.
The detection results of multiple batches of samples show that the compound arisaema analgesic plaster contains mesaconine, aconitine and hypo-aconitineThe content of aconitine is 3.2-5.0 mug/g respectively-1、0.9～1.3μg·g-1、8.5～10.4μg·g-1Within the range, the content determination result of the hypaconitine in the preparation is basically consistent with the HPLC determination result, which shows that the 2D-UHPLC detection method has better applicability and can be used for the quality control of products. The research result provides a useful reference for qualitative and quantitative research of trace components in complex components or complex matrixes of traditional Chinese medicines, and also provides data support for clinical safety and reasonable medication.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (5)
1. A method for detecting diester alkaloids in compound arisaema analgesic plaster is characterized by comprising the following steps:
step one, preparing a test solution, namely extracting and removing impurities from a compound arisaema analgesic plaster sample to prepare the test solution; step two, absorbing a test solution, and determining diester alkaloids by a two-dimensional liquid chromatography method, wherein the diester alkaloids are selected from mesaconitine, aconitine and hypaconitine;
the two-dimensional liquid chromatography method comprises one-dimensional chromatography and two-dimensional chromatography, wherein the one-dimensional chromatography is carried out by taking methanol-0.001-0.02% of diethylamine or triethylamine as a mobile phase system, and the two-dimensional chromatography is carried out by taking acetonitrile-0.01% of phosphoric acid as a mobile phase system;
a one-dimensional chromatographic method: c18The first specification of the chromatographic column is 2.1 × 50mm, 1.7 μm, and the flow rate is 0.1 mL.min-1Column temperature 18 ℃; gradient elution, 0-12 min, 40-58% A, 12-25 min, 58-58% A, 25-35 min, 58-65% A, 35-40 min, 65-70% A, 40-42 min, 70-70% A, 42-45 min, 70-100% A, 45-48 min, 100-40% A;
the valve switching method comprises the following steps: the valve switching time is from the initial peak output time to the end of the diester alkaloid;
two-dimensional chromatographic methods: c18The second specification of the chromatographic column is 3.0 × 100mm, 1.7 μm, and the flow rate is 0.7 mL.min-1The column temperature is 40 ℃; gradient elution, 0-1 min, 30-38% A, 1-3 min, 38-48% A, 3-4 min, 48-90% A, 4-4.2 min and 90-30% A; the detection wavelength was 235 nm.
2. The detection method according to claim 1, wherein the detection wavelength is 235nm, the amount of the sample is 1. mu.L, and the temperature of the sample plate is 10 ℃.
3. The detection method according to claim 1, wherein C is18Chromatography column one and said C18The second chromatographic column is selected from Waters Xbridge BEH C18A chromatographic column.
4. The detection method according to claim 1, characterized in that: the extraction and impurity removal in the first step are as follows: adding appropriate amount of methanol into the product, grinding, quantitatively transferring to a measuring flask, ultrasonic treating, diluting with methanol to scale, shaking, filtering, discarding the primary filtrate, collecting the subsequent filtrate, and recovering under reduced pressure to dry; adding hydrochloric acid solution, slightly heating to dissolve the residue, shaking, refrigerating in refrigerator, filtering, collecting the filtrate, placing in a separating funnel, extracting with chloroform, mixing chloroform extractive solutions, dehydrating with anhydrous sodium carbonate, recovering the filtrate under reduced pressure to dryness, dissolving the residue with methanol, metering volume, shaking, and centrifuging.
5. Use of the assay method as defined in claim 1 for quality control of analgesic paste of Arisaema cum bile.
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